1. Field of the Invention
The present invention relates generally to the field of signal transduction, transcriptional regulation and bone physiology. More specifically, the present invention relates to the role by which Smad6 interacts with nuclear Hox proteins during bone morphogenetic protein (BMP) signal transduction.
2. Description of the Related Art
Members of TGF-β superfamily transduce their signals into the cell through a family of mediator proteins called Smads. Receptor-regulated Smad1, Smad5 and Smad8 mediate BMP signaling, whereas Smad2 and Smad3 respond to TGF-β (9–12). Upon phosphorylation by their type I receptors, the receptor-regulated Smads interact with a common partner, Smad4, and translocate to the nucleus where the complex recruits DNA binding protein(s) to activate specific gene transcription (1,2,13–15).
Smad6 and Smad7 are struturally divergent Smads and antagonists of TGF-β family signaling (1). Smad6 and Smad7 are characterized by the stable interactions formed with both activated TGF-β and BMP type I receptors, thereby preventing phosphorylation of ligand-induced Smads (4,5). In addition, Smad6 has also been demonstrated to interact with phosphorylated Smad1 to prevent the formation of an active signaling complex of Smad1 and Smad4, preferentially inhibiting the signaling pathways activated by bone morphogenetic proteins (7,16). Furthermore, it was previously demonstrated that Smad1 interacts with Hoxc-8 in response to BMP stimulation (13). Hoxc-8 functions as a transcriptional repressor. The interaction of Smad1 with Hoxc-8 dislodges Hoxc-8 binding from its element, thereby resulting in initiation of gene transcription (13).
In vertebrates, there are 39 Hox homeobox-containing transcription factor genes, organized into four separate chromosome clusters, which play critical roles in the process and patterning of vertebrate embryonic development (28,29). These 39 genes are subdivided into 13 paralogous groups on the basis of duplication of an ancestral homeobox cluster during evolution, sequence similarity and position within the cluster (30). Each paralog group has been demonstrated to be responsible for morphogenesis of a particular embryonic domain or structure (29). There are three members in Hox paralog group VIII, Hoxb-8, Hoxc-8 and Hoxd-8 (30). Hox genes are required during vertebrate limb bud development, and particularly, Hoxb-8 was suggested to be a transcription factor involved in activating the Sonic hedgehog gene, which is the key mediator in limb development (31,32). Furthermore, Northern blot analysis shows that Hoxc-8 is expressed during human embryo development at high levels in spinal cord, backbone and limbs and at a lower level in heart (33). BMP-2/4 activates expression of Hox genes, induces osteoblast differentiation and controls patterning across the anteroposterior (a-p) axis of developing limb (34).
The prior art is deficient in recognizing the role of Smad6 in conjunction with transcriptional regulation by Hox genes. The present invention fulfills this long-standing need and desire in the art and further provides methods of gene regulation and screening for drugs using the teachings of the present invention.